Small Molecule Cancer Treatments that Cause Necrosis in Cancer Cells But Do Not Affect Normal Cells

ABSTRACT

A method of treating cancer in a subject, including: providing a subject having a plurality of cancer cells; and administering to the subject, a therapeutically effective amount of a composition including: an HDM-2 binding component; and a membrane resident component, the membrane resident component bound to the HDM-2 binding component. Also provided are a method of selectively necrosing cancer cells, a method of causing membranolysis in cancer cells, and a cancer treatment composition.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/193,437 filed 5 Mar. 2021, which is a continuation of U.S. patentapplication Ser. No. 16/661,036 filed 23 Oct. 2019, which is acontinuation of U.S. patent application Ser. No. 15/372,229 filed 7 Dec.2016, which is a continuation of U.S. patent application Ser. No.12/744,831 filed on 9 Dec. 2010, which was granted as U.S. Pat. No.9,539,327, which is the U.S. National Phase of International PatentApplication No. PCT/US2008/084810 filed 26 Nov. 2008, which claimspriority to U.S. Provisional Patent Application No. 60/990,276 filed 26Nov. 2007, all of which are incorporated herein by reference in theirentirety.

FUNDING STATEMENT

The present invention was made possible by funding from the AmericanCollege of Surgeons Faculty Fellowship Research Award and awardidentifier USPHS NIH CA 42500, and from the Lustgarten Foundation forPancreatic Cancer Research. The government may have certain rights tothe invention.

FIELD OF THE INVENTION

The present invention is directed to an HDM-2-targeting cancertreatment. Specifically, the present invention is directed to methods ofvarious cancer treatments that target HDM-2 in cancer cells, killingcancer cells by necrosis while not affecting normal, non-cancerouscells.

BACKGROUND

New approaches to cancer treatments are needed to yield effective andefficient cancer treatments. Some cancer treatments are directed to thep53 mechanism within cells. The p53 protein blocks the oncogenic effectsof a number of oncogenic proteins that induce mitosis. Absence of thep53 protein is associated with cell transformation and malignantdisease.

Cancer treatments which target the p53 protein within the cancer cellshave been developed recently. However, some types of cancer cells do nothave p53, while others exhibit p53 in a mutated, inactive form. Thus,these p53 targeting cancer treatments are ineffective at treatingcancers as these treatments do not have any effect on cells withinoperative (mutant) or nonexistent p53. Also, normal non-cancerouscells have p53, so it is unclear what detrimental affects thesetreatments may have on non-cancerous cells.

SUMMARY OF THE INVENTION

In an aspect of the present invention, a method of treating cancer in asubject is provided. The method includes: providing a subject having aplurality of cancer cells; and administering to the subject, atherapeutically effective amount of a composition including: an HDM-2binding component; and a membrane resident component (or membraneresident peptide, ‘MRP’), the membrane resident component bound to theHDM-2 binding component.

In another aspect of the present invention, a method of selectivelynecrosing cells is provided. The method includes: providing a pluralityof cells, including at least one cancer cell and at least one normalnon-cancerous cell; administering to the cells a composition, where thecomposition includes an HDM-2 binding component and a membrane residentcomponent, the membrane resident component bound to the HDM-2 bindingcomponent; where the composition results in membranolysis of one or moreof the cancer cells, but does not affect the normal non-cancerous cells.

In still another aspect of the present invention, a method of causingmembranolysis in cancer cells is provided. The method includes:administering to at least one cancer cell a compound including an HDM-2binding component and a membrane resident component, the membraneresident component bound to the HDM-2 binding component.

In yet another aspect of the present invention, a cancer treatmentcomposition is provided. The composition includes: an HDM-2 bindingcomponent; and a membrane resident component, where the membraneresident component is attached to the HDM-2 binding component, where thecomposition causes selective membranolysis when administered to a sampleof cells, the sample containing a plurality of cancer cells and aplurality of normal non-cancerous cells, further where membranolysisonly occurs in the cancer cells.

The embodiments of the present invention may be better understoodthrough a study of the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a method of treating cancer in a subject.

FIG. 2 depicts a method of selectively necrosing cells.

FIG. 3 depicts a method of causing membranolysis in cancer cells.

FIG. 4 is a table outlining various cancer cell lines to which PNC-27 orPNC-28 were administered, further depicting the time to total celldeath.

FIG. 5 is a table depicting that various untransformed cell lines andprimary human cells were unaffected when incubated with PNC-27 for oneto several days.

FIGS. 6A-6B depicts two transmission electron microscopy (TEM)photographs showing the isolated plasma membrane fractions of BMRPA1(6A, on the left-hand side) and BMRPA1.TUC3 (6B, on the right-hand side)Mag.×85,000. Such TEM analyses were performed to monitor plasma membranepurification for the immunoblot (IB) studies summarized in FIG. 7 .

FIGS. 7A-7B depicts the IB data of cell lysates (7A) and plasmamembranes (7B) fractions for MDM-2 (90-95 kDa) and p53 of untransformed(rodent primary pancreatic acinar BMRPA1 cells) and cancer cells (mouseB16 Melanoma, rodent pancreatic cancer BMRPA1.TUC3, and human pancreaticcancer MIA PaCA-2 cells).

FIG. 8 is a TEM photograph (Mag.×46,200) of one human pancreatic cancercell (MIA-PaCa-2 cell line) taken 10 minutes after PNC-28administration. The top arrow depicts a pore formed in the cancer cellmembrane, the middle arrow depicts an eruption of the cellular membranein response to the PNC-28, while the bottom arrow depicts adiscontinuous region in the cellular membrane as a result of the PNC-28administration.

FIGS. 9A-9D depict scanning electron microscope (SEM) images of humanpancreatic cancer cells (MiaPaCa-2) that have not been treated withcancer treatments; the cell surfaces are depicted as intact and smooth.

FIGS. 10A-10D depict SEM images of human pancreatic cancer cells(MiaPaCa-2) to which PNC-27 has been administered for 3 min at 37° C.);note the granular surface and pores (arrows) formed on the surface ofthe cancer cell membranes.

FIG. 11 depicts a schematic of the PrecisionShuttle subcloningprocedure. The entry and destination vectors are digested with SgfI andMluI, which rarely cut in mammalian coding sequences. After a ligationreaction, the resulting clones are grown on ampicillin-containing mediumto select for successful subclongin of the ORF into the destinationvector.

FIG. 12 depicts the experimental results of an MTT assay taken fortransfection experiments.

FIGS. 13A-13D graphs A through D depict the results of dose-responseexperiments completed on three difference cancer cell samples and innormal primary human fibroblasts to treatment in vitro with PNC-27.

DETAILED DESCRIPTION

This invention relates to the surprising discovery by the presentinventors of the selective mechanism of action of certain peptides; thatwhen administered to cancer cells and normal non-cancerous cells,necrosis of cancer cells occurs, but the normal non-cancerous cells areunaffected. This surprising result led to the invention of the novelmethods to treat cancer, and compositions of the present invention fortreating the same. More specifically, this invention involves methods oftreatment and compositions of synthetic peptide, non-peptide, andcombination molecules for treating cancer, where the compoundsselectively destroy malignant and transformed cells only, even whenadministered to a mixture of normal non-cancerous cells and cancercells. Also included are methods of treating cancer.

As is known, the p53 protein is a vital regulator of the cell cycle. Itblocks the oncogenic effects of a number of oncogene proteins thatinduce mitosis, in part by blocking transcription of proteins thatinduce mitosis and by inducing the transcription of proteins that blockmitosis, and promotes apoptosis. Absence of the p53 protein isassociated with cell transformation and malignant disease. Haffner, R &Oren, M. (1995) Curr. Opin. Genet. Dev. 5: 84-90.

The p53 protein molecule consists of 393 amino acids. It includesdomains that bind to specific sequences of DNA in a DNA-binding domainthat consists of residues 93-312. The crystal structure of this regionhas been determined by x-ray crystallography. Residues 312-393 areinvolved in the formation of homotetramers of the p53 protein. Residues1-93 constitute the trans-activating domain and are involved inregulation of the activity and half life of the p53 protein.

The gene encoding the p53 protein is that is gene most commonlydisrupted in cancer. p53 protein acts as the guardian of the genome, asit guards against copying of DNA. It was previously established that thep53 gene within cells was a target treatment for cancer. However, p53targeting treatment in cancer cells has various problems associated withit that limits the use of p53 targeting treatments. For example, not allcancers exhibit p53 in the cell. Targeting treatments for these types ofcancers would not work, as there is no p53 for the targeting compoundsto bind to. Also, some cancers exhibit a mutated form of p53, which isinactive. As mutant p53 is inactive in these cancers, targetingcompounds also do not work for these cancers. Thus, p53 dependenttreatment mechanisms are ineffective against these types of cancer.

The p53 protein binds to another important regulatory protein, the HDM-2protein. As used herein, “MDM-2” refers to the regulatory protein inmice, “RDM-2” refers to the regulatory protein in rats, while “HDM-2”refers to the regulatory protein in humans. MDM-2 and HDM-2 havesubstantially similar roles in cancer cells, so experiments related tothe function and role of the protein may be completed with either humancells (studying HDM-2), rat cells (studying RDM-2), or mouse cells(MDM-2). The HDM-gene that encodes the HDM-2 protein is a knownoncogene. The HDM-2 protein forms a complex with the p53 protein, whichresults in the degradation of the p53 protein by a ubiquitinationpathway. The p53 protein binds to HDM-2 protein using an amino acid (AA)sequence that includes residues 12-26 of the p53 protein, which areinvariant. The entire HDM-2 protein binding domain of the p53 proteinspans residues 12-26. Haffner, R & Oren, M. (1995) Curr. Opin. Genet.Dev. 5: 84-90. The HDM-2 protein is the expression product of a knownoncogene, so the HDM-2 protein is a very important regulatory protein.

Some cancer treatments have been developed that utilize molecules toblock the formation of the complex between the p53 protein and the MDM-2protein that causes the inhibition of transcriptional activity of thep53 protein. Thus the anti-tumor effect of these molecules prlong thehalf-life of wild-type p53 enhancing its anti-tumor activity. Moregenerally, these and other experimental observations have beeninterpreted as suggesting that the anti-tumor effect of the p53 proteinmight be enhanced by peptides capable of interfering with the binding ofthe MDM-2 protein to the p53 protein which is thought to result in anextension of the life-span (t/2) of p53 and in an increase in thecellular level of p53. Indeed, a number of investigators have suggestedthat the MDM-2/p53 complex might be a target for rational drug design.See, e.g., Christine Wasylyk et al., “p53 Mediated Death of CellsOverexpressing MDM-2 by an Inhibitor of MDM-2 Interaction with p53”,Oncogene, 18, 1921-34 (1999), and U.S. Pat. No. 5,770,377 to Picksley etal.

The inventors of the present invention have surprisingly discovered thatcancer cells have roughly five times as much HDM-2 in their cellmembranes as do normal non-cancerous cells. This novel discovery has ledthe present inventors to design new compositions and methods of treatingcancer that focus on and take advantage of the newly discoveredknowledge related to HDM-2.

As used herein, cancer includes any disease or disorder associated withuncontrolled cellular proliferation, survival, growth, or motility.Cancers that may be treated or prevented by the present inventioninclude any cancer whose cells have increased expression of HDM-2 intheir plasma membranes. Such cancers may include, for example,pancreatic cancer, breast cancer, colon cancer, gastric cancer, prostatecancer, thyroid cancer, ovarian cancer, endometrial cancer,glioblastoma, astrocytoma, renal carcinoma, lung cancer, sarcoma,including osteogenic sarcoma, mesothelioma, sporadic nonfamilial tumors,lymphoma, and others including hematologic cancers such chronicmyelogenous leukemia. Precancerous conditions, where cells exhibit highamounts of HDM-2 in the plasma membrane, are also included as treatablewith the compositions and methods of the present invention.

Specifically, the inventors have designed novel non-p53 cancertreatments that focus on the characteristics of cancer cells in atargeted cancer cell treatment. One aspect of the composition includes amembrane transport component, which is quickly and effectivelytransported through the cancer cell membrane. The other aspect of thecomposition includes an HDM-2 binding component, which targets HDM-2 inthe cell membrane, and binds to the HDM-2 therein. The two componentsare attached, (e.g., chemically bound to each other), so that, while themembrane resident component (e.g. the membrane resident peptide (orMRP)) inserts into the cancer cell membrane, the HDM-2 binding componentattaches to the HDM-2 in the cancer cell membrane. This interactionbetween the HDM-2-binding segment and HDM-2 in the membrane holds thepeptide in the membrane in which it adopts a membrane-activeconformation allowing it to form pores in the cancer cell membrane. Thisresults in rapid tumor cell necrosis. A similar phenomenon occurs at themitochondrial membrane within the cancer cell where significant amountsof HDM-2 are also present.

Thus, the administration of the compositions to one or more cancer cellsresults in the formation of pores in the cell membranes of the cancercells. The presence of the membrane resident peptide on the end of theHDM-2 binding component allows the peptide to become membrane-active andto form well-defined pores in the cell membrane, causing membranolysis,which allow for extrusion of the intracellular contents from theinterior of the cancer cell, resulting in the compromise of theintegrity of the cell. Pores in the cancer cell membrane are formed asan immediate result of administration of the compound. After the poresare formed, cell necrosis, or cell death, results within a short timeframe, ranging from 15 minutes to 48 hours. Pore formation in the plasmamembrane, in turn, causes membranolysis of the cancer cells, as theholes in the cell walls start allowing excess fluid and free compound torush into the cells which, simultaneously, start leaking cytoplasm intothe surrounding environment. The compound entering the cells through theplasma membrane pores binds to the HDM-2 and forming pores in themitochondrial membrane causing lysis of the mitochondria and the abrupttermination of cellular energy production. Once membranolysis starts,the cell eventually undergoes necrosis, or cell death, as a result ofthe treatment with the methods and compositions of the presentinvention.

Therefore, the compositions and methods of treatment have devastatingeffects on cancer cells. Normal, non-cancerous cells are unaffected, asnormal cells have from none to negligible amounts of HDM-2 in theirplasma membranes as compared to cancer cell membranes. Even if thecomposition is transported into normal cells (as through membranetransport of the membrane resident component), the composition has nomeasurable effect on the normal, non-cancerous cells.

Thus, not only do the methods and compositions of the present inventiontend to eradicate cancer cells, they may be administered to cancer cellsand healthy cells alike, and only cancer cells will be affected.Further, as the proposed mechanism of action is non-p53 targeting, thesemethods and compounds will be effective in the treatment of morevarieties of cancer, including those cancers which have no p53 present,or an inactive p53 as the result of mutation. Additionally, thesetreatments may be effective against cancers which, due to their locationin the body, may otherwise be considered undetectable or inoperable.Thus, the methods and compositions of the present invention solve theproblems associated with p53 targeting treatments and provide aneffective and efficient treatment of cancer cells in a subject,preferably a mammal, more preferably a human.

The materials and methods of the present invention provide novel methodsof treatment that are directed to the newly discovered commoncharacteristic of various forms of cancer, the presence of HDM-2 in thecancer cell membrane. Such materials and compositions may be used as ageneral treatment to many forms of cancer, which, up until now, may havevery different treatment options and/or varying degrees of success.

Though cancers may not exhibit similar characteristics pathologically orphysiologically within subjects diagnosed therewith, or typicaltreatment avenues, many of these cancers, though admittedly different,have high levels of HDM-2 present in their cell membranes. Cancers thathave been identified as having a large amount of HDM-2 in the cellmembrane include, for example: MIA-PaCa-2 human pancreatic cancer cells,BMRPA1.TUC-3 rat pancreatic cancer cells, MCF-7 human breast cancercells, B16 mouse melanoma, and a human melanoma A2025 cells.

The compositions of the present invention have both an HDM-2 bindingdomain and a membrane resident component (or membrane resident peptide),thus, the compositions have selectivity and a high affinity for cancercells, and will thus only bind to and cause necrosis of cancer cellswhen administered to a combination of cancer cells and normal, healthycells. These new methods and compositions provide effective treatments,screening methods for additional novel drug candidates, and otherbenefits and advantages over the current state of the art.

The compositions of the present invention which are employable with themethods of the present invention include generally an HDM-2 binding siteand a membrane resident component, or membrane resident peptide.Molecules that bind to HDM-2 may be composed of residues that are sharedby the p53 HDM-2 binding domain, or may alternatively, be smallmolecules with a tendency or high affinity for binding to HDM-2.

Thus, these methods may be used to treat a sample of cells containingboth non-cancerous, normal cells and cancer cells. Such samples wouldinclude cell lines, tissue samples, tumors, and/or a subject havingcancer in need of treatment. As the methods of treatment do not causecell death of normal cells, these methods of treatment are focused onthe cancer cells, irrespective of the mode of administration to the cellsample. Thus, these methods of treatment may be used for tumors orcancers that are widespread, inoperable, or otherwise not effectivelytreated with conventional means or combination therapies.

The present invention provides methods of using peptides whichcorrespond to all or a portion of amino acid residues 12-26 of human p53protein. When fused to a membrane resident peptide (or non-peptide,membrane resident component) the peptides are lethal to malignant ortransformed cells. The subject peptides are thus useful in treatingcancer in an animal, preferably a human.

The compositions of the present invention may include, for example,PNC-27 and PNC-28, as disclosed and described in pending U.S. patentapplication Ser. No. 11/977,521, filed on Oct. 25, 2007 (U.S.Publication No. 20080076713), and Ser. No. 11/582,687, filed on Oct. 26,2006 (U.S. Publication No. 20070238666), the contents of both of theseapplications are incorporated by reference herein in their entireties.

Additionally, one or more compositions may be used, where a compound mayhave both shared residues of p53 as an HDM-2 binding domain, and alsomembrane resident component. The inventors of the present invention havedeveloped a peptide from the HDM-2-binding domain of p53 attached to amembrane resident peptide (or component) sequence that causes necrosis,not apoptosis, of tumor, but not normal, non-cancerous cells. Thepeptides include both PNC-27 and PNC-28, which are p53-derived peptidesfrom the human double minute binding domain (HDM-2) that are attached tothe membrane resident peptide (MRP). These synthetic peptides inducecell necrosis of cancer cells, but not normal non-cancerous cells. Theanti-cancer activity and mechanism of PNC-27 (p53 aa12-26-MRP) andPNC-28 (p53 AA 17-26-MRP) were specifically studied by the inventors ofthe present invention as against human pancreatic cancer, though usesand applications are included with the various methods of the presentinvention.

Preferably, the membrane resident peptide, which, besides beingnecessary for cell membrane insertion, stabilize includes predominantlypositively charged amino acid residues since a positively chargedsequence, as it stabilizes the alpha helix of a subject peptide or smallmolecule component. Examples of MRPs that may be employed are referencedin U.S. patent application Ser. No. 11/997,521 filed on Oct. 25, 2007,published as United States Patent Application Publication No.20080076713, the contents of which are incorporated herein by referencein its entirety. Additional examples of MRP sequences which may belinked to the HDM-2 binding peptides of the present invention aredescribed in Futaki, S. et al (2001) Arginine-Rich Peptides, J. Biol.Chem. 276, 5836-5840.

It should be noted that p53 targeting cancer treatments and HDM-2targeting cancer treatments cause cell death through different modes. Asis known, cell death can occur through either necrosis or apoptosis.p53-targeting treatments typically cause cell death through apoptosis,while the embodiment features of the present invention cause cell deathby necrosis. Necrosis is uncontrolled cell death, while apoptosis isgenetically controlled or “programmed” cell death. Apoptosis is thedeliberate cellular response to specific environmental and developmentalstimuli or programmed cell death. Cells undergoing apoptosis exhibitcell shrinkage, membrane blebbing, chromatin condensation andfragmentation. In contrast, necrosis involves the destruction ofcytoplasmic organelles and a loss of plasma membrane integrity.Apoptosis of cancer cells by p53 targeting treatments fails to treatthose cancers that do not exhibit p53, or, through mutations, exhibit aninactive p53 form that is unresponsive to p53 targeted treatments.

Referring to FIG. 1 , a method 100 of treating cancer in a subject isprovided. The method 100 includes: providing 110 a subject having aplurality of cancer cells; and administering 120 to the subject, atherapeutically effective amount of a composition including: an HDM-2binding component; and a membrane resident component, the membraneresident component bound to the HDM-2 binding component. The method mayfurther comprise the step of observing a physiological result 134. Asreferenced herein, observing a physiological result may refer toobserving a change to one or more cancer cells and/or observing a changein a living subject. Changes may be observed through changes in cellcharacteristics, including pore formation on the plasma membrane ofcancer cells. Further, observations may be made, for example, forextrusion from the treated cancer cell of an increased amount of lactatedehydrogenase (LDH), a cytosolic enzyme, in the cellular medium and/orthe observation of necrosis in cells. One or more assays and/orexperiments may be completed in order to observe a result of the method100 of the present invention.

Referring to FIG. 2 , a method 200 of selectively necrosing cells isprovided. The method includes: providing 112 a plurality of cells,including at least one cancer cell and at least one normal non-cancerouscell; administering 120 to the cells a composition, wherein thecomposition includes an HDM-2 binding component and a membrane residentcomponent, the membrane resident component bound to the HDM-2 bindingcomponent; wherein the composition results in membranolysis of thecancer cells, but does not affect the normal non-cancerous cells. Thestep of providing 112 a plurality of cells may be, for example,providing at least one cell line, providing a cell sample, tissuesample, tumor sample, or a live subject having cancer. The method 200refers to “selectively necrosis” as only the cancer cells from a mixtureof cancer cells and healthy cells, will be killed, or necrotized.

Referring to FIG. 3 , a method 300 of causing membranolysis in cancercells is depicted. The method includes: administering 120 to at leastone cancer cell a compound including an HDM-2 binding component and amembrane resident component, the membrane resident component bound tothe HDM-2 binding component. The method 300 may further include the stepof observing at least one result thereof 130. Observable results mayinclude observing the results of the administration 120 step, as throughvisual inspection, or microscopic inspection.

Further, the method 300, as well as the methods 100 and 200, may includethe step of measuring at least one result 140. Measuring 140, as usedherein, indicates that more than mere passive observation is completed.Measuring 140 may encompass administering various assays and analyticaltests to ascertain or analyze one or more results of the administrationstep. Such analytical tests and assays are described herein.

It should also be noted that one or more of the steps of the methods100, 200, 300, as set forth herein, may be repeated or reiterated 150until a desired result is reached. A desired result may be a certainpercentage of necrosis in the cancer cells, a reduced tumor size in asubject, and the like. Repeated a reiterated 150 administration may, forexample, include a treatment regimen or therapy designed by a clinicianor a doctor.

The step of providing 110 as set forth within methods 100, 200 and 300,may include, for example, providing a living subject diagnosed withcancer, suspected of having cancer, refractory, or relapse, or a subjectwho has been transplanted or xenotransplanted with cancerous cells. Theplurality of cells, as referenced herein, may refer to a cell, a cellline, a plurality of cells in vivo, a combination of two or more typesof cells (i.e. cancerous and non-cancerous cells), a tumor, more thanone tumor, and the like.

A composition with “activity” as a cancer treatment with reference tothe embodiments of the present invention refers to an ability to inducea desirable effect upon in vitro, ex vivo, or in vivo administration ofthe compound. Desirable effects include preventing or reducing thelikelihood (increasing the likelihood or causing) one or more of thefollowing events: binding to HDM-2 in cancer cells, insertion into thecancer cells' plasma membrane, assembly and pore foundation, transportacross the cancer cell membrane, causing membranolysis.

The terms “therapeutically effective dosage” and “effective amount”refer to an amount sufficient to kill one or more cancer cells. Atherapeutic response may be any response that a user (e.g. a clinicianwill recognize) exhibits as an effective response to the therapy,including the foregoing symptoms and surrogate clinical markers. Thus, atherapeutic response will generally be an amelioration or inhibition ofone or more symptoms of a disease or disorder, e.g. cancer.

The term subject, as used herein may refer to a patient or patientpopulation diagnosed with, or at risk of developing one or more forms ofcancer. Also, as used herein, a subject may refer to a living animal,including mammals, which may be given cancer through transplantation orxenotransplanting which may be subsequently treated with the methods andcompounds of the present invention or which have developed cancer andneed veterinary treatment. Such subjects may include mammals, forexample, laboratory animals, such as mice, rats, and other rodents;monkeys, baboons, and other primates, etc. They may also includehousehold pets or other animals in need of treatments for cancer.

Further to the methods 100, 200, and/or 300, the administration step 120may be done through various forms, as is known. Administration of thesynthetic peptides of the present invention may be by oral, intravenous,intra-arterial, intranasal, suppository, intraperitoneal, intramuscular,intradermal or subcutaneous administration or by infusion orimplantation. When administered in such manner, the synthetic peptidesof the present invention may be combined with other ingredients, such ascarriers and/or adjuvants. There are no limitations on the nature of theother ingredients, except that they are preferably pharmaceuticallyacceptable, efficacious for their intended administration, preferably donot degrade the activity of the active ingredients of the compositions,and preferably do not impede importation of a subject peptide into acell. The compounds may also be impregnated into transdermal patches, orcontained in subcutaneous inserts, preferably in a liquid or semi-liquidform which patch or insert time-releases therapeutically effectiveamounts of one or more of the subject synthetic compounds.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. The ultimatesolution form in all cases must be sterile and fluid. Typical carriersinclude a solvent or dispersion medium containing, e.g., water bufferedaqueous solutions, i.e., biocompatible buffers, ethanol, polyols such asglycerol, propylene glycol, polyethylene glycol, suitable mixturesthereof, surfactants or vegetable oils. Sterilization may beaccomplished utilizing any art-recognized technique, including but notlimited to filtration or addition of antibacterial or antifungal agents.

Administering may include contacting. The term “contacting” refers todirectly or indirectly bringing the cell and the compound together inphysical proximity. The contacting may be performed in vitro or in vivo.For example, the cell may be contacted with the compound by deliveringthe compound into the cell through known techniques, such asmicroinjection into the tumor directly, injecting the compound into thebloodstream of a mammal, and incubating the cell in a medium thatincludes the compound.

The compounds of the invention are administered to a human in an amounteffective in achieving its purpose. The effective amount of the compoundto be administered can be readily determined by those skilled in theart, for example, during pre-clinical trials and clinical trials, bymethods familiar to physicians and clinicians. Typical daily dosesinclude approximately 1 mg to 1000 mg.

Any method known to those in the art for contacting a cell, organ ortissue with a compound may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vitro methods typically includecultured samples. For example, a cell can be placed in a reservoir(e.g., tissue culture dish), and incubated with a compound underappropriate conditions suitable for inducing necrosis in cancer cells.Suitable incubation conditions can be readily determined by thoseskilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the compound under appropriate conditions.The contacted cells, organs or tissues are normally returned to thedonor, placed in a recipient, or stored for future use. Thus, thecompound is generally in a pharmaceutically acceptable carrier.

In vivo methods are typically limited to the administration of acompound, such as those described above, to a mammal, preferably ahuman. The compounds useful in the methods of the present invention areadministered to a mammal in an amount effective in necrosing cancercells for treating cancer in a mammal. The effective amount isdetermined during pre-clinical trials and clinical trials by methodsfamiliar to physicians and clinicians.

An effective amount of a compound useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thecompound may be administered systemically or locally.

The compounds useful in the methods of the invention may also beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

Any formulation known in the art of pharmacy is suitable foradministration of the compounds useful in the methods of the presentinvention. For oral administration, liquid or solid formulations may beused. Some examples of formulations include tablets, capsules, such asgelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The compounds can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

Formulations of the compounds useful in the methods of the presentinventions may utilize conventional diluents, carriers, or excipientsetc., such as those known in the art to deliver the compounds. Forexample, the formulations may comprise one or more of the following: astabilizer, a surfactant, preferably a nonionic surfactant, andoptionally a salt and/or a buffering agent. The compound may bedelivered in the form of an aqueous solution, or in a lyophilized form.Similarly, salts or buffering agents may be used with the compound.

Formulations and Administrations

The compounds of the present invention may be administered intherapeutically effective concentrations, to be provided to a subject instandard formulations, and may include any pharmaceutically acceptableadditives, such as excipients, lubricants, diluents, flavorants,colorants, buffers, and disintegrants. Standard formulations are wellknown in the art. See, e.g. Remington's pharmaceutical Sciences, 20^(th)edition, Mach Publishing Company, 2000. The formulation may be producedin useful dosage units for administration by any route that will permitthe compound to contact the cancer cell membranes. Exemplary routes ofadministration include oral, parenteral, transmucosal, intranasal,insulfation, or transdermal routes. Parenteral routes includeintravenous, intra-arterial, intramuscular, intradermal, subcutaneous,intraperitoneal, intraductal, intraventricular, intrathecal, andintracranial administrations.

The compounds of the present invention may be administered as a solid orliquid oral dosage form, e.g. tablet, capsule, or liquid preparation.The compounds may also be administered by injection, as a bolusinjection or as a continuous infusion. The compounds may also beadministered as a depot preparation, as by implantation or byintramuscular injection.

It should also be noted that necrosis of cells generally triggers animmunologic response in the body of a subject, often resulting ininflammation and/or swelling. As such, the methods of the presentinvention 100, 200, and 300, may also include the step of administeringan anti-inflammatory agent or medicament. Many commonly acceptedanti-inflammatory agents may be used, as is known in the art.

Yet another embodiment of the present invention provides a cancertreatment composition. The composition includes: an HDM-2 bindingcomponent; and a membrane resident component, wherein the membraneresident component is attached to the HDM-2 binding component, whereinthe composition causes selective membranolysis when administered to asample of cells, the sample containing a plurality of cancer cells and aplurality of normal non-cancerous cells, further wherein membranolysisoccurs only in the cancer cells.

The compounds and methods 100, 200, 300 of the present invention may beadmixed or otherwise combined with a pharmaceutically acceptablecarrier. As used herein, a “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic agents and the like. The use of suchmedia and agents are well-known in the art.

The phase ‘pharmaceutically acceptable’ refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce unwanted reactions when administered to a subject, particularlyhumans. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term carrier refers to a diluent, adjuvant, excipient orvehicle with which the compounds may be administered to facilitatedelivery. Such pharmaceutical carriers can be sterile liquids, such aswater and oils, or organic compounds. Water or aqueous solution salinesolutions, and aqueous dextrose and glycerol solutions are preferablyemployed as carriers, particularly as injectable solutions.

The synthetic peptides of the present invention may be synthesized by anumber of known techniques. For example, the peptides may be preparedusing the solid-phase technique initially described by Merrifield (1963)in J. Am. Chem. Soc. 85:2149-2154. Other peptide synthesis techniquesmay be found in M. Bodanszky et al. Peptide Synthesis, John Wiley andSons, 2d Ed., (1976) and other references readily available to thoseskilled in the art. A summary of polypeptide synthesis techniques may befound in J. Stuart and J. S. Young, Solid Phase Peptide Synthesis,Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also besynthesized by solid phase or solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath, H. et al., Eds., pp. 105-237,Academic Press, New York, N.Y. (1976). Appropriate protective groups foruse in different peptide syntheses are described in the texts listedabove as well as in J. F. W. McOmie, Protective Groups in OrganicChemistry, Plenum Press, New York, N.Y. (1973). The peptides of thepresent invention may also be prepared by chemical or enzymatic cleavagefrom larger portions of the p53 protein or from the full length p53protein. Likewise, membrane-resident sequences for use in the syntheticpeptides of the present invention may be prepared by chemical orenzymatic cleavage from larger portions or the full length proteins fromwhich such leader sequences are derived.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject peptide selectively lethalto malignant and transformed mammalian cells. The present invention alsocontemplates a deoxyribonucleic acid (DNA) molecule that defines a genecoding for, i.e., capable of expressing a subject peptide or a chimericpeptide from which a peptide of the present invention may beenzymatically or chemically cleaved.

When applied to cells grown in culture, synthetic peptides areselectively lethal to malignant or transformed cells, resulting in adose-dependent reduction in the cell number. The effect is observablegenerally within two to three and at most 48 hours.

One or more of the methods 100, 200, 300 may further include the step ofdetermining whether a compound reduces cancer cells activity furtherincludes measuring the level of lactate dehydrogenase (LDH) in the cellmedium, observing the cells for pore formation or membranolysis, orobserving the cells breaking down over a period of time. The level ofnecrosis may be measured by any method known in the art, including forexample, PCR analysis, RT-PCR, Northern blot, Western blot,immunohistochemistry, ELISA assays, luciferase reporter assays, etc.

One or more of the methods of the present invention may be repeated orreiterated 150 on a subject. This may be desirable, for example, if asubject suffers from refractory or relapse cancer. Also, repeatedadministration may be desirable if lower dosages are administeredrepeatedly, over a treatment cycle or pursuant to combination therapy.

Identifying drug candidates typically involves multiple phases. Duringthe early stages, compounds, preferably large libraries of compounds arescreened or tested in vitro for binding to and/or biological activity atthe cancer cell membrane (with HDM-2 and/or a membrane residentcomponent characteristic). The compounds that exhibit activity (“activecompounds” or “hits”) from this initial screening process are thentested through a series of other in vitro and in vivo tests to furthercharacterize the anti-cancer normal, non-cancerous tissue and organprotective activity of the compounds.

The in vivo tests at this phase may include tests in non-human mammalssuch as those mentioned above. If a compound meets the standards forcontinued development as a drug following in vitro and in vivo tests,the compound is typically selected for testing in humans.

A progressively smaller number of test compounds at each stage areselected for testing in the next stage. The series of tests eventuallyleads to one or a few drug candidates being selected to proceed totesting in human clinical trials. The human clinical trials may includestudies in a human suffering from a medical condition that can betreated or prevented by reducing cancer cells (inducing cancer cellnecrosis).

The compounds and methods 100, 200, 300 of the present invention may bedesigned to have one or more desirable characteristics. The desirablecharacteristics may result in an increased effectiveness when thecomposition is administered to at least one cancer cell or in vivo to anorganism, particularly a mammal, in need of cancer treatment.

Desirably, compounds and compositions of the present invention may havea three dimensional shape or conformation in analpha-helix-loop-alpha-helix. This is the three-dimensional shape thathas been determined by the present inventors for the PNC-27 and PNC-28peptide-based compositions. The alpha-helix-loop-helix conformationallows the composition to advantageously interact with the cancer cellmembrane.

It may also be desirable for the compositions of the present inventionto be of a higher degree of rigidity than the synthetic peptides PNC-27and PNC-28. As is known, peptide-based compositions have naturalmovement associated with their molecules. As discussed, thealpha-helix-loop-alpha-helix, that results in an amphipathic structure,in which hydrophobic amino acid residues occupy one face of the moleculewhile polar residues occupy the opposite face of the molecule, is adesired conformation of the molecule. A number of membrane-activepeptides, such as melittin and magainin, have these required structuresthat result in cell membrane lysis though not with the same specificityas PNC-27. Thus, if agents can be administered to a peptide-basedcomposition to increase the rigidity, or if a non-peptide, called apeptidomimetic, rigid molecules of similar size, with a similaramphipathic structure, may be employed with the present invention, thenthe conformation will more immediately affect the cancer cells. Rosal R,Brandt-Rauf P W, Pincus M R, Wang H, Mao Y, Fine R L. The role ofalpha-helical structure in p53 peptides as a determinant for theirmechanism of cell death: necrosis versus apoptosis. Adv Drug Deliv Rev2005; 57:653-60; Pincus, M. R. (2001) “The Physiological Structure andFunction of Proteins” in Principles of Cell Physiology (Chapter 2),Third Edition, Ed., N. Sperelakis, Academic Press, New York, pp. 19-42;3. Dathe, M. and Wieprecht, T. (1999) Structural Features of HelicalAnti-Microbial Peptides: Their Potential to Modulate Activity on ModelMembranes and Biological Cells. Biochem. Biochem. Biophys. Acta 1462,71-87.

Another desirable characteristic is for a relatively small-sizedcomposition to be employed with the methods and as the compositions ofthe present invention. Large peptide, non-peptide, and combinationpeptide/non-peptide compositions have the disadvantage of triggering animmunogelogic response with a greater likelihood than small molecules,which may go unnoticed in vivo. Thus, the immune system of the organismbeing treated is less likely to trigger an immune response against smallmolecules, i.e. peptides of <35AA than large molecule compositions,i.e., proteins with >35AA. Preferably, the synthetic peptide materialsof the present invention are on the order of about thirty-five (35)amino acids or fewer.

Generally, as molecules (proteins) exceed 5000 D(˜>35AA in size, theybecome more immunogenic, i.e., they can elicit an immune response in therecipient. Peptides up to 5000 D (<35 AA) have been found to elicit noor only a minor immune response in the recipient. However, long-term(many months) application of peptides from 2500 D to 5000 D can resultin stimulating an immune response (they can become immunogenic) in somerecipients. Considering the size of our peptides (27AA and 32AA or withLeupeptin 30AA and 35AA) and of the long-life constructs describedbelow, they are all in the non- to borderline-immunogenic range. Takinginto account (1) that all PNC-peptides, including those with—leupeptinattached, will have a rather short lifespan (estimates are 10 to 30 min)due to removal by pinocytosis degradation, and (2) that they are appliedto tumor-bearing patients most of which are immunologically suppressed,the likelihood of developing immunological responses that will restricttheir use is very, very remote.

Yet another desirable characteristic for the composition is to have along half-life. A composition with a long half-life is able to stay inthe body for longer periods of time before decomposing. Thus, acomposition with a longer half-life may have an increased longevity,allowing it to be transported through the body to kill more cancer cellsor treat cancers located in different parts of the organism upon asingle administration. Peptide-based compounds, including PNC-27 andPNC-28, may be altered to include a D-amino acid on the amino terminalend in order to slow peptidase activity of the molecule. Similarly,leupeptin, a known peptidase activity inhibitor, may be attached to thecarboxyl terminal end of PNC-27 and PNC-28 in order to slow peptidaseactivity and lengthen the half-life of the molecules. The syntheticpeptides of employed with the methods of the present invention areprobably likely to have half-lives on the order of minutes in situ, asis the case for most therapeutic peptides.

To the cancer cell lines depicted in FIG. 4 , either PNC-27 or PNC-28 invarying doses were administered to the cancer cells. Cancer cell linestested include: Rat Pancreatic cancer, Rat Brain Angiosarcoma, MouseMelanoma, Cervical Squamous Cell Cancer, Non-small cell Lung Cancer(both with p53 and p53 null), Human Breast Cancer (including p53present, p53 mutant, and p53 null), Human Melanoma, Human PancreaticCancer, and Human Ovarian Cancer. To each sample, doses ranging from 60microMolar to 75 milliMolar were administered to the cancer cells. Totalcell death, in cell samples 1×10⁶ cells, occurred in these cell samplesas soon as 30 minutes up to about 72 hours. Even with low dosagesadministered in this experiment, cancer cells were eradicated in a veryswift timeframe. This data tends to suggest that lower dosages may beadministered to exhibit a therapeutic result. While all doses ultimatelykill all of the cancer cells, lower doses tend to take longer to killthe cancer cells than higher doses. For example, at doses of 125 μg/mland higher, the times vary from 15 minutes to 72 hours, depending on thecancer cell line.

The experimental methods which yielded the data depicted in FIG. 5 aredescribed in sufficient detail for those skilled in the art in thefollowing publications:

-   Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F.    K., Rubinstein, P., Visser, I., Robinson, R., Brandt-Rauf, P. W.,    Michl, J., Fine, R. L. and Pincus, M. R. (2001) Peptides from the    Amino Terminal mdm-2 Binding Domain of p53, Designed from    Conformational Analysis, Are Selectively Cytotoxic to Transformed    Cells. Proc. Natl. Acad. Sci. USA 98, 12438-12443;-   Do, T. N., Rosal, R. V., Drew, L., Raffo, A. J., Michl, J.,    Pincus, M. R., Friedman, F. K., Petrylak, D. P., Cassai, N.,    Szmulewicz, J., Sidhu, G., Fine, R. L. and Brandt-Rauf, P. W. (2003)    Preferential Induction of Necrosis in Human Breast Cancer Cells by a    p53 Peptide Derived from the mdm-2 Binding Site. Oncogene 22,    1431-1444; and-   Bowne, W. B., Sookraj, K. A., Vishnevetsky, M., Adler, V., Yadzi,    E., Lou, S., Koenke, J., Shteyler, V., Ikram, K., Harding, M.,    Bluth, M. H., Ng, M., Brandt-Rauf, P. W., Hannan, R., Bradhu. S.,    Zenilman, M., Michl, J. and Pincus, M. r. (2008) The Penetratin    Sequence in the Anti-Cancer PNC-28 Peptide Causes Tumor Necrosis    Rather than Apoptosis of Human Pancreatic Cancer Cells. Ann. Surg.    Oncol., in press.

Referring to FIG. 5 , the table illustrates that the cancer treatmentPNC-27, when administered to non-cancerous cells, has no effect. ThirtymicroMolar dosages were administered to cell types including: normal ratpancreatic acinar cells, human breast cells, primary human fibroblasts,primary human keratinocytes, and umbilical cord stem cells (taken fromfive donors). The cells were incubated with the PNC-28 or PNC-27respectively, synthetic peptide for various time frames ranging from oneday up to two weeks, with no measurable effect on the non-cancerouscells. This data supports the conclusion of the inventors of the presentinvention, that the methods and compounds of the present invention havean immediate affect upon cancer cells, causing necrosis in a very shorttime frame, while having no measurable effect on non-cancerous, healthycells.

FIG. 6 depicts the transmission electron microscopy results of theplasma membrane fractions of BMRPA1 (normal rat pancreatic acinar cells)and BMRPA1.TUC3 cells (rat pancreatic cancer-human oncogenick-ras^(val12)). The arrows point to the classic structure of the lipidbilayer of the many plasma membrane sheets and vesicles that have beencaptured in the two photographs. The photographs demonstrate thesignificant enrichment of plasma membrane that the purification hasachieved. Nevertheless, additional cellular materials are enclosed inthe newly formed, mostly inside-out membrane vesicles. Now that we haveisolated the plasma membrane fraction of cancer cells and normal cells,we have performed immunoblotting of the plasma membrane proteins andtotal cell lysates for MDM-2, HDM-2 and p53 as shown in FIG. 7 .(Magn.×85,000).

FIG. 7 shows that MDM-2 and p53 are found, as expected, in the wholecell lysates, while the plasma membrane fractions clearly demonstratethat p53 is not present in the cell membrane of either cancer or normaluntransformed cells. In contrast, MDM-2 and HDM-2 are present in thecell membrane of cancer cells, but not in the plasma membrane ofuntransformed normal cells. Of specific importance in this context isthat while whole cell lysates of normal untransformed BMRPA1 cellsclearly contain MDM-2 and p53 protein, the cells' plasma membrane arecompletely devoid of the two proteins. In contrast, the plasma membraneof their BMRPA1.TUC3 daughter cells that had been transformed to cancercells by direct transfection of oncogenic human k-ras^(val12) containssignificant amounts of MDM-2. The absence of p53 in these and the plasmamembrane preparations of the other cancer cells further indicates thatMDM2 or HDM-2 (human cancer cells) expressed in the cancer cells' plasmamembrane is not complexed to p53.

FIG. 8 depicts a photograph taken during a Transmission ElectronMicroscopy study of human pancreatic cancer MIA PaCa-2 cells that hadbeen treated for 10 minutes with PNC-28. The top arrow depicts an areain which a needle-shaped pore has formed in the cancer cell plasmamembrane. The middle arrow depicts an area of the cell's plasma membranethat has erupted as a result of PNC-28 acting upon it. The bottom-mostarrow depicts an area on the cell's plasma membrane that isdiscontinuous as a result of PNC-28 administration. From one or more ofthese areas emphasized with arrows, the cytoplasm will begin to leakfrom the cell, into the surrounding medium. Extrusion of much of thecells cytoplasmic content through such holes (pores) while the nuclearmembrane was still intact was a frequent finding in these and othertumor cells when the incubation period with PNC-28 was extended.Magn.×42,600.

FIGS. 9 and 10 show the before and after PNC-27 treatment images ofhuman pancreatic cancer MIA PaCa-2 cells as observed by scanningelectron microscopy (SEM).

In FIG. 9 (A-D), the cell surfaces of the untreated human pancreaticcancer MIA PaCa-2 cells are depicted as relatively continuous andsmooth. The appearance of the untreated human pancreatic cancer MIAPaCa-2 cells as shown in FIG. 9 is in stark contrast with the humanpancreatic cancer MIA PaCa-2 cell images of FIG. 10 , which have beentreated with PNC-27 for 3 min at 37° C. Referring to FIG. 10 , A and B,the human pancreatic cancer MIA PaCa-2 cells have a granular appearanceon their surfaces. This is the PNC-27 that has gathered on and about thecancer cell membrane, presumably in complex with HDM-2.

As is shown in FIG. 10 , C and D, dark discontinuous regions, or pores,have been formed in the cancer cell membranes. For ease in reference,some of the pores in FIG. 10 C and D are emphasized with arrows pointingtowards the pore. Also, about many of the pores, there are gatherings orclumps of PNC-27. This tends to suggest that not only is PNC-27 formingpores in the cancer cell membranes, but also that PNC-27 that has notbecome membrane active may enter the cancer cell through the poresformed by other PNC-27 molecules. (Magnifications are evident by thebars.)

Since PNC-27 has an HDM-2 binding domain and since HDM-2 occurs in themembranes of cancer cells but normal cells and since PNC-27 isselectively toxic to cancer cells but not normal cells, we concludedthat PNC-27 binds to HDM-2 in the cancer cell membrane. We performed twosets of experiments to prove this conclusion. In the first set ofexperiments, in the first set of experiments, we performedco-localization studies. To show analytically that PNC-27 co-localizeswith HDM-2, in the plasma cell membrane, experiments were performed withfluorescent-labeled antibodies.

Cancer cells were treated with PNC-27 at a dose of 125 ug/ml (its IC₅₀)or lower concentrations, like 25 ug/ml, that will allow binding of thepeptide to HDM-2 but will not rapidly kill the cells. After the cellswere treated, the cells were incubated first with the anti-p53 antibodythat recognizes p53 residues 12-26. This antibody is called DO-1, and itis labeled with FITC, a green fluorescent probe. The cells were washedfree of any excess antibody. Then the cells were incubated with ananti-HDM-2 antibody that was been conjugated to a red fluorescent dye.After washing the cells free of excess antibody, the cells weresubjected to confocal microscopy. We found that the red and greenfluorescence occurred together, in the cell membrane only, giving awell-defined yellow color (combination of red and green) rather thanseparate red and green fluorescence's. This showed that PNC-27co-localizes with HDM-2 in the cancer cell membrane. For the control,cancer cells were incubated with FITC-labeled DO-1 (the anti-p53antibody that recognizes the p53 12-26 sequence that is part of PNC-27).Then, confocal microscopy was used to show that there is no fluorescenceon the cell membrane. This rules out the possibility that p53 alsooccurs in the cell membrane. Therefore the only cause for greenfluorescence in cancer cells treated with PNC-27 and then DO-1 is thepresence of PNC-27 in the membrane.

In a second set of experiments, we showed that we could make normalcells susceptible to PNC-27 if we induced expression of HDM-2 in theirplasma membranes. The manner in which we accomplished this was totransfect a plasmid into normal MCF-10-2A breast epithelial cells thatare not susceptible to PNC-27. This plasmid encoded the full lengthHDM-2 protein attached to a CAAX sequence (Cys-Ile-Leu-Lys) that targetsnewly synthesized HDM-2 to the plasma membrane. We also transfected anumber of control plasmids into these cells. Overall, each plasmid hadthe following principal structure as shown in FIG. 11 .

Referring to FIG. 11 , the depiction provides a schematic of the plasmidcalled PrecisionShuttle for subcloning procedure is provided (obtainedfrom OriGene, Rockville, MD). The entry and destination vectors aredigested with Sgf and Mlu I, which rarely cut in mammalian codingsequences. After a ligation reaction, the resulting clones are grown onampicillin-containing medium to select for successful subcloning of theORF into the destination vector. From 11:00 O'clock, going clockwise:TAG=tag protein, in this case, green fluorescent protein (GFP), SgfI,endonuclease restriction site; protein ORF=HDM-2 sequence; MLU1=endonuclease restriction site; the next darker gray segment is justplasmid DNA; neo=neomycin resistance gene (black); Amp′=ampicillinresistance gene (gray segment). Next is the black segment, also plasmidDNA at the end of which is the promoter that causes expression of bothGFP and HDM-2 to be constitutive.

The following HDM-2 constructs were made: 1. Full length HDM-2; 2. Fulllength HDM-2 with the sequence Cys-Ile-Leu-Lys, (CAAX sequence) thatcauses the whole protein to be inserted into the cell membrane; 3.Partial length HDM-2 that lacks residues 1-109 (containing the p53- andPNC-27-binding domain), with the sequence Cys-Ile-Leu-Lys, (CAAXsequence), called del-1-109-HDM-2-CAAX that causes the whole protein tobe inserted into the cell membrane; and 4. Empty vector that does notexpress HDM-2 at all. Based on the expression of GFP, the transfectionof each of the constructs into MCF-10-2A was successful.

With the transfected constructs, two experiments were completed. Theexperiments included confocal microscopy measure of HDM-2 expression,pre- and post-administration of PNC-27. In the latter experiment, thecells were incubated with PNC-27. They were then incubated with aprimary antibody against PNC-27, called DO-1. The cells were washed andincubated with an antibody to HDM-2. They were washed again. The cellswere then incubated with fluorescent secondary antibodies to each of theprimary antibodies to PNC-27 and HDM-2. The red fluorescent antibody wasto PNC-27 while the green fluorescent antibody was to HDM-2. The cellswere incubated with PNC-27 and tested for their viabilities using theMTT assay.

There were several possible patterns. If HDM-2 is expressed in the cellmembrane and binds to PNC-27, both red and green fluorescence shouldappear together, which appear as visible to the observer as a yellowcolor. If PNC-27 inserts into the membrane and HDM-2 is expressed in themembrane, but there is no interaction (as we anticipate withdel-1-109-HDM-2-CAAX), separate and distinct red and green fluorescenceshould be visibly observable, but no yellow fluorescence. If HDM-2 isnot expressed in the cell membrane, there should be no greenfluorescence in the membrane, but only inside the cell. If PNC-27 staysin the cell membrane, punctuate red fluorescence in the cell membraneshould be observable.

We found that empty vector-transfected cells had no green (HDM-2) intheir membranes and very little red (PNC-27) anywhere. The transfectionof the full-length construct with membrane-attaching sequence (CAAX)resulted in yellow observable in the membrane showing co-localization ofPNC-27 with HDM-2. The truncated del-1-109-HDM-2-CAAX-containing cellsshowed separate red and green fluorescence after treatment with PNC-27suggesting that PNC-27 does not bind to truncated HDM-2. Cellstransfected with full-length HDM-2, without the membrane localizationsignal showed no green fluorescence (HDM-2) in the cell membrane butonly in the cytosol. This illustrates that HDM-2 is not expressed in thecell membrane of normal non-cancerous cells. Red fluorescence (PNC-27)is seen in the membrane only. Thus there is no co-localization of PNC-27and HDM-2 in the membrane (no yellow in the membrane).

FIG. 12 depicts the MTT assay results for cell viability for theMCF-10-2A human breast epithelial cells that have been transfected witheach respective construct and then incubated with PNC-27. Each constructvector is labeled on the X-axis with the viability result (%proliferation) on the Y-axis. The dark bar-graphs are for PNC-27experiments while the unfilled bar graphs are for a control peptide thathas no effect on any cells, cancer or normal non-cancerous cells, calledPNC-29. Note that cell viabilities are very similar between PNC-27- andcontrol peptide-treated cells except for MDM-2-CAAX, which isfull-length MDM-2 attached to CAAX. These cells are killed by PNC-27.These are the only cells that show that PNC-27 co-localizes withH(M)DM-2-CAAX in the cell membrane. This shows that it is HDM-2expressed in the cancer cell membrane that is responsible for thespecificity of PNC-27. Since we have shown that PNC-27 induces selectivecancer cell necrosis by a mechanism that involves its binding to HDM-2in the cancer cell membrane, PNC-27-induced cancer cell killing will bemore likely show a strong dose dependence.

FIG. 13 , graphs A through D, depict the results of dose-responseexperiments completed on three difference cancer cell samples and onenormal primary human cell line to treatment in vitro with PNC-27 (from1-500 micrograms/ml). The cancer cell lines were human pancreaticcancer, MiaPaCa-2 (upper left, A), Hu-Melanoma cells (upper right, B),and a rodent tumor cell line, i.e., pancreatic cancer BMRPA1.TUC-3 cells(lower left, C). As controls, cells from the same cell lines were testedin parallel with a control peptide, PNC-29, over the same range ofpeptide concentrations. As another control, cells from primary human Ag13145 fibroblasts (lower right, D) were treated with the same doses ofPNC-27 and PNC-29, respectively.

The results are plotted along with those for PNC-29, the negativecontrol. The results indicate that (1) there is a strong dose dependencyas expected, (2) the effective range is different for different tumorcells, which have different (LD₅₀), (3) there is absence of any effectover the same dose range of a control peptide, and (4) PNC-27 has noeffect on healthy primary human fibroblasts when used over the same doserange as applied to tumor cells. The measurements of cytotoxicity wereperformed by assaying for LDH in the cell supernatant. PrototypicalSmall Molecules that Exhibit the Characteristics of PNC-27 that AreSelectively Cytotoxic to Cancer Cells

PNC-27 and PNC-28 are synthetic peptides which include an HDM-2 bindingregion and a membrane resident peptide. Various tests andexperimentation completed by the present inventors have led theinventors to the surprising discovery that not only peptide, but alsocombined peptide and non-peptide, as well as wholly non-peptidematerials may be constructed to exhibit and induce selective andspecific cancer cell-necrosis, while leaving normal non-cancerous cellsunaffected.

Once the present inventors surprisingly determined the role of eachcomponent of the synthetic peptide molecules, it became possible todesign additional molecules based on the discoveries of the proposedmechanism of action. Because peptide-based materials arecharacteristically non-rigid, the synthetic peptides employed with themethods of the present invention may require some time in order to adoptthe proper conformation before they can bind to their desired target(here, HDM-2). Also, compositions that are not wholly composed ofpeptides may exhibit longer half lives, upon administration to aplurality of cancer cells or in vivo after administration to a livingorganism with cancer. Further, the size of newly designed compositionsare in accordance with the tested synthetic peptides, as sizes shorterthan 32 AA can typically be administered to a living organism whilerarely triggering an immune response.

As such, hybrid materials containing peptide and non-peptide components,along with wholly non-peptide materials may be used with the methods ofthe present invention. The synthesis of one or more of the compounds maybe subsequently followed by purification, as is commonly done in theart. The compounds synthesized are preferably in purified form to beused as the compound and with the methods of the present invention.

Purified form, as used herein, generally refers to material which hasbeen isolated under certain desirable conditions that reduce oreliminate unrelated materials, i.e. contaminants. Substantially freefrom contaminants generally refers to free from contaminants withinanalytical testing and administration of the material. Preferably,purified material is substantially free of contaminants is at least 50%pure, more preferably, at least 90% pure, and more preferably still atleast 99% pure. Purity can be evaluated by conventional means, e.g.chromatography, gel electrophoresis, immunoassay, composition analysis,biological assay, NMR, and other methods known in the art.

It should be noted that though the below-referenced experiments have notyet been completed on the other compounds useable with the methods ofthe present invention, the inventors contemplate similar results asobtained herein, based on the characteristics of the various componentsof the compounds, in combination with the proposed and verifiedmechanism of action.

TABLE I Compositions of the Present Invention and Employable with theMethods of the Present Invention No HDM-2 binding component MembraneResident Component Name 1 12-26 p53 protein, residues (PPLSQETFSDLWKLL)KKWKMRRNQFWVKVQRG PNC-27 (SEQ ID NO: 1) (SEQ ID NO: 3) 2 17-26 p53protein, residues (ETFSDLWKLL) KKWKMRRNQFWVKVQRG PNC-28 (SEQ ID NO: 2)(SEQ ID NO: 3) 3 12-26 p53 protein, residues (P*P*LSQETFSDLWKLL)KKWKMRRNQFWVKVQRG PNC-27-D (SEQ ID NO: 1), where * denotes D-amino acid,(SEQ ID NO: 3) amino acid rather than natural, L-amino acid (D&L areoptical isomers) 4 12-26 p53 protein, residues (PPLSQETFSDLWKLL)KKWKMRRNQFWVKVQRG- PNC-27- (SEQ ID NO: 1) LLR (SEQ ID NO: 3) leupeptin 517-26 p53 protein, residues KKWKMRRNQFWVKVQRG- PNC-28- (ETFSDLWKLL) (SEQID NO: 2) LLR (SEQ ID NO: 3) leupeptin 6

KKWKMRRNQFWVKVQRG (SEQ ID NO: 3) Nutlin-2- MRP 7

Guanidinylated biphenyl (structure shown below) Nutlin-2- guanidinylatedbiphenyl 8 12-26 p53 protein, residues (PPLSQETFSDLWKLL) Guanidinylatedbiphenyl p5312-26 (SEQ ID NO: 1) (structure shown below) guanidinylatedbiphyenls 9 XFMXXXEXLX, where X in first position is actylKKWKMRRNQFWVKVQRG Non-native moiety (CHO), X in fourth position isalpha-amino- (SEQ ID NO: 3) p53-MRP isobutyric acid, X in fifth positionis phosphonomethyl- phenylalanine, X in sixth position is6-chlorotryptophan, X in eighth position is1-amino-cyclopropanecarboxylic acid, X in tenth position is NH2. (SEQ IDNO. 10)

TABLE II SEQUENCE ID NOS: SEQUENCE ID NO. SEQUENCE Name SEQ ID NO: 1PPLSQETFSDLWKLL Residues 12-26 p53, MDM-2 binding domain SEQ ID NO: 2ETFSDLWKLL Residues 17-26 p53, MDM-2 binding domain SEQ ID NO: 3KKWKMRRNQFWVKVQRG Membrane resident peptide (MRP), AntennapediaSEQ ID NO: 4 MPFSTGKRIMLGE Sequence from cytochrome P450,used as a control SEQ ID NO: 5 PPLSQETFSDLWKLLKKWKM PNC-27 RRNQFWVKVQRGSEQ ID NO: 6 ETFSDLWKLLKKWKMRRNQF PNC-28 WVKVQRG SEQ ID NO: 7MPFSTGKRIMLGEKKWKMRR PNC-29 (Control NQFWVKVQRG Peptide) SEQ ID NO: 8PPLSQETFSDLWKLLKKWKM PNC-27-Leupeptin RRNQFWVKVQRGLLRX, whereX is acetyl moiety SEQ ID NO: 9 ETFSDLWKLLKKWKMRRNQF PNC-28-LeupeptinWVKVQRGLLRX, where X is Acetyl moiety, CHO SEQ ID NO: 10XFMXXXEXLX, where X in first Non-native p53position is actyl moiety (CHO), X [Ac-Phe-Met-Aib-in fourth position is alpha-amino- pmp-6-Cl-Trp-isobutyric acid, X in fifth position Glu-Ac₃c-Leu-NH₂,is phosphonomethyl- where phenylalanine, X in sixth positionAib = alpha-amino- is 6-chlorotryptophan, X in eighth isobutyric acid,position is 1-amino- pmp = phosphonometcyclopropanecarboxylic acid, X in hyl-phenylalanine,tenth position is NH2. 6-Cl-Trp = 6- chlorotryptophan, Ac₃c = 1-amino-cyclopropanecarboxylic acid.] SEQ ID NO: 11LLRX, where X is acetyl moiety, Leupeptin CHO

PNC-27-D Amino Acid

In order to slow the degradation of the PNC-27 synthetic peptide insitu, it may be desirable to attach a D-amino acid to the p53 part ofthe protein. The D-amino acid may be inserted into the peptide chain, orpreferably exchanged, such that one of the existing amino acids may takethe form of its optical isomer, ‘D’ form, over the naturally occurring‘L’ form. The ‘D’ amino acid may be part of the originally synthesizedpeptide. Preferably, this may be an amino acid towards the aminoterminal end. This may best be accomplished by adding one amino acid onthe amino terminus of the peptide, e.g., D-alanine. The D amino acid maybe synthetically placed on the synthetic peptides through the use ofmethods known to those skilled in the art. The conformation and helicityof the synthetic peptide with at least one D-amino acid added to theamino terminus of the parent peptide is not expected to disrupt theactive three-dimensional structure of the peptide. This can be confirmedin solution studies such as by Nuclear Magnetic Resonance (NMR).

PNC-27-Leupeptin or PNC-28-Leupeptin

In order to lengthen the half life of the synthetic peptide material,leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) (LLRX, where X is theacetyl moiety)(SEQ ID NO:11), a well-known protease inhibitor may beattached to the synthetic peptide. As both PNC-27 and PNC-28 are smallpeptides, upon administration they may be quickly catabolized ordegraded by extra- and intracellular proteases. Thus, methods known tothose skilled in the art may be used to attach leupeptin, a knownprotease inhibitor, with capabilities to inhibit a broad spectrum ofproteases, onto the synthetic peptides. For example, leupeptin may besynthesized onto the end of the MRP by solid state synthesis, a commonmethod for creating synthetic proteins. Also, leupeptin may bechemically added onto the carboxyl terminal end of the MRP byconventional chemical means, as known to those skilled in the art.

Preferably, the leupeptin is attached to the membrane resident peptidein such a manner that once the MRP is inserted into the cancer cellmembrane, the leupeptin is able to disassociate and inhibit proteaseactivity inside and outside the cell. Alternatively, leupeptin mayremain attached and may bind to nearby proteases, blocking theirdegradation of the peptide. Thus, leupeptin may desirably be split offfrom MRP to inhibit protease in the cell. Thus, the synthetic peptidePNC-27 or PNC-28, with leupeptin attached to the carboxyl terminal endof the MRP will likely considerably lengthen the life span of thesynthetic peptide in situ.

It is desired that leupeptin is attached to or towards the carboxylterminal end of the membrane resident component. Attachment to thecarboxyl rather than the amino terminal end is due to the fact that thearginine residue must be maintained in the aldehyde state; as oxidationto the carboxyl oxidation state results in inactivity. Also withleupeptin on the carboxyl terminus, no serious impact to the threedimensional structure of the overall peptide is contemplated to becreated. In fact, since it is positively charged, it will likely furtherstabilize the desired alpha-helical conformation of the MRP and of theHDM-2 binding component. The addition of leupeptin to PNC-27 or PNC-28is believed to maintain the conformation of the MRP while increasing thehalf life of the synthetic peptide material. Thus, the compound (PNC-27or PNC-28, with leupeptin attached to its carboxyl terminal end) mayremain active in the body of the subject for a longer period of time. Itshould be noted that since leupeptin is a small peptide composed ofthree amino acids (leucine-leucine-arginine), the addition of leupeptinonto the carboxyl terminal end of the MRP will likely not greatlyincrease the size of the synthetic peptide.

Methods known and used by those skilled in the art, including NMR,spectroscopy, and/or computational modeling, may be used to analyze andconfirm the structure and effects of the synthetic peptide attached toleupeptin. For a detailed discussion of leupeptin and the family ofsimilar small molecule enzyme (protease) inhibitors which may belikewise incorporated as compounds employable with the presentinvention, the following publications are provided:

-   Moldoveanu, T., et al., Crystal Structures of Calpain-E64 and    -Leupeptin Inhibitor Complexes Reveal mobile Loops Gating the Active    Site, J. Mol. Biol. (2004) 343, 1313-1326    (doi:10.1016/j/jmb.2004.09.016); and-   Rohr, K. et al., X-ray Structures of Free and Leupeptin-complexed    Human αI-Tryptase Mutants: indication for an α→β-Tryptase    Transition, J. Mol. Biol. (2006) 357, 195-209    (doi:10.1016/j.jmb.2005.12.037). The aforementioned publications are    incorporated herein by reference in their entireties.

Nutlin-2-MRP

Nutlins are known to those in the art as small molecule compounds shownto have an MDM-2 binding affinity. Structure, uses, and characteristicsof Nutlins are known by those in the art and are disclosed and describedin several references, including:

-   Vassilev, L. T., et al., In-Vivo Activation of the p53 Pathway by    Small-Molecule Antagonists of MDM-2, SCIENCE, v. 303, 6 Feb. 2004,    844-848 (www.sciencemag.org); and-   Vassilev, L. T., et al, Selective small-molecule inhibitor reveals    critical mitotic functions of human CDK1, PNAS, v. 103, No. 28, Jul.    11, 2006, 10660-10665. The contents of both of these articles are    incorporated herein by reference. Nutlins act on cancer cells to    block the p53-hdm-2 interaction. Nutlins come in three different    forms, nutlin-1, nutlin-2, and nutlin-3. However, for the purposes    of this invention, nutlin-2 may be employed. Its structure is shown    below.

Preferably, the inventors of the present invention attach nutlin-2 tothe amino terminal end of the membrane resident peptide (MRP) to createa compound of the present invention and which may be administered inaccordance with the methods 100, 200, 300 of the present invention.Nutlin-2 contains a desirable reactive group, i.e., the —CH₂OH group,which may be the site for further chemistry to attach the nutlin-2 tothe MRP.

In this case, nutlin-2 is esterified to the side chain —COOH group ofterminally blocked aspartic acid; this becomes the first modified aminoacid in the MRP. The nutlin-2 component has a known MDM-2/HDM-2 bindingaffinity, while the membrane resident component has a membrane activecharacter which is disclosed and described herein.

Nutlin-2-Guanidinylated Biphenyl

Small molecule carriers are known by those skilled in the art, and aredisclosed and described in the publication: Okuyama, M. and Laman, H.and Kingsbury, S. R. and Visintin, C. and Leo, E. and Edward, K. L. andStoeber, K. and Boshoff, C. and Williams, G. H. and Selwood, D. L.Small-molecule mimics of an α-helix for efficient transport of proteinsinto cells. Nature Methods, 4 (2). pp. 153-159 (2007). ISSN 15487091,which is incorporated by reference herein in its entirety.

The nutlin-2-guanidinylated biphenyl compound may be used as theanti-cancer compound or with one or more of the methods 100, 200, 300 ofthe present invention in order to cause membranolysis in cancer cells.As a non-peptide peptidomimetic agent, this compound may exhibit alonger half-life than the synthetic peptide materials when administeredto a subject or to a plurality of cells in situ.

The biphenyls were designed to contain a reactive amino group to which asuccinic acid moiety was attached, leaving a very reactive —COOH groupthat allows attachment of a wide variety of compounds. Nutlin-2 is idealin that it has a reactive —CH2OH group as shown in the two figuresabove. This can readily be esterified to the —COOH of the succinatemoiety of the guanidinylated biphenyl.

Certain experiments may be desirably completed on the syntheticnon-peptide nutlin-2-guanidinylated biphenyl material in order toanalyze and confirm its characteristics as an anti-cancer activity. Forexample, one million cancer cells are to be incubated with a dose rangeof the new compound (nutlin-2-guanidinylated biphenyl) from 10 nM to 100uM and cell viability may be analyzed (as set forth for the PNC-27 andPNC-28 peptides above) using trypan blue exclusion and MTT assay,analyzed for the release of LDH into the medium, and analyzed forcaspase-3 (for possible apoptosis).

In the in vitro experiments with PNC-27 and PNC-28, the peptides wereadded with new medium every one-two days at the specified dose whentotal cell killing occurs over several days. When the cancer cells aretreated with a peptidomimetic, it will not be necessary to add morecompound periodically because the half-life of the peptidomimetic isproposed to be much greater than for the parent peptide. The inventorsexpect with the aforementioned experiments, that the medium will notneed to be changed to incorporate additional compound. Thus, one dose ofthe peptidomimetic may be sufficient total cancer cell killing.

P53-12-26-Guanidinylated Biphenyls

Another compound of the present invention includes the p53 12-26residues chemically bound to the guanidinylated biphenyl material,previously disclosed and described above. In order to attach theguanidinylated biphenyl to the HDM-2 binding component (residues 12-26of p53), the inventors of the present invention will take terminallyblocked serine and react it with an activator of its side chain-OHgroup. Next, the inventors will add the biphenyl, which contains thesuccinic acid moiety. This will form a covalent ester bond withterminally blocked serine. Then, this modified terminally blocked serinewill be added to the solid-phase synthesized p53 residues 12-26, to addit onto the activated carboxyl terminal end of this peptide on the solidphase column. This will give us p53 12-26 peptide linked toguanidinylated biphenyl-serine. The synthesized guanidinylated biphenylsattached to the carboxyl terminal end of the MDM-2 binding domain (p53residues 12-26) may exhibit a longer half life than the purely peptidematerials as this compound lacks the MRP peptide residues that can behydrolyzed. Also the presence of the “abnormal” carboxyl terminal serinemay inhibit peptidases. This may also contribute to a lower IC₅₀ forthis compound as compared to the purely peptide materials, PNC-27 andPNC-28.

The fact that we have a helical, positively charged moiety on thecarboxyl terminal end of the p53 12-26 sequence makes it very likelythat this hybrid molecule will be very active, so it will bind to hdm-2while the non-peptide biphenyl moiety will may it amphipathic, membraneactive and allow pore formation. In order to further analyze and verifythe structure of the compound, molecular modeling and/ormulti-dimensional NMR studies in solution may be completed.

Non-Native p53-MRP

Non-native p53, as referred to herein, refers toAc-Phe-Met-Aib-pmp-6-Cl-Trp-Glu-Ac₃c-Leu-NH₂, WhereAib=alpha-amino-isobutyric acid, pmp=phosphonomethyl-phenylalanine,6-Cl-Trp=6-chlorotryptophan, Ac₃c=1-amino-cyclopropanecarboxylic acid(XFMXXXEXLX, where X in first position is actyl moiety (CHO), X infourth position is alpha-amino-isobutyric acid, X in fifth position isphosphonomethyl-phenylalanine, X in sixth position is6-chlorotryptophan, X in eighth position is1-amino-cyclopropanecarboxylic acid, X in tenth position is NH2.)(SEQ IDNO: 10). The synthesis and structure of this compound is known to thoseskilled in the art, as it is publicly available in the publications:Chene, P. et al (2000) J. Mol. Biol.; Chene, P. et al (2002) FEBS Lett.529, 293-297. In these references, non-native p53 materials were usedfor blocking the binding of p53 to HDM-2 in cancer cells. In furtheranceto the compound and methods 100, 200, 300 of the present invention,non-native p53 may be attached to the MRP through straight solid phasesynthesis, as is known to those skilled in the art. Similarly, achemical complexing step may be completed in order to bind thenon-native p53 to the MRP. In the case of the unnatural amino acids,since the carboxyl terminal residue is a leucine, a fusion of theunnatural sequence to amino terminal activated MRP can be performed.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or else where in the specification, to provide additionalguidelines to the practitioner in describing the compositions andmethods of the invention, as well as how to make and use them.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. Patents, patent application, publications,products descriptions, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entries for all purposes.

Experimental Protocol:

The following provides a discussion of the experimental methods employedfor various experimental results described herein.

Confocal Microscopy:

Cells were released with trypsin from their TCFs and grown on glasscover slips in 24-well dishes until they reached 50-60% density. Afterremoval of the spent medium and PBS washing buffer they were treated forup to 15 min at 37° C. in a humidified 5% CO₂—95% air incubator chamberwith PNC-27 or PNC-29 (control) at 50 μg/ml incubation medium. At theend of the incubation the cells were washed and fixed in 3%paraformaldehyde in PBS (pH 7.2) supplemented with 0.01% glutaraldehydefor 1.5 h followed by extensive washing and transfer into PBS forstorage until mounting on glass slides for microscopy. Free aldehydegroups were quenched by incubating cells with glycine and sodiumborohydride (NaBH₄), followed by washing in PBS. Cells were then stained(direct staining) for 2 h, 4° C., with fluorescein-labeled mousemonoclonal antibody against p53 [FITC-mAbα-p53 (DO-1)] (5 μg/ml) andrhodamine-labeled (TRITC-) mAbα-against H/R/MDM-2 (5 μg/ml). Afterremoval of non-reactive Ab and extensive washing, the cover slips weremounted over antifade (Molecular Probes—Invitrogen, CA) on glass slidesand examined with a laser-equipped Olympus Confocal microscope 1×76.Results were digitally recorded. The co-localization of the two Abs wasconfirmed by overlapping green (anti-p53) and red (anti-H/R/MDM-2)fluorescent labels which produced a yellow color.

Transmission Electron Microscopy:

The following is the experimental protocol for the TEM picture shown inFIG. 6 . Cells (2×10⁶) were grown in 75 cm² TCFs until ˜80% confluencywhen they were placed on ice, washed with ice cold PBS, scraped andcollected in PBS, and centrifuged at 500×g, for 10 min and at 50° C.After removal of the supernatant the cell pellet was resuspended inhomogenization buffer [0.022M Na—PO₄, pH7.4, 0.001M MgCl₂, 0.25Msucrose, cocktail of protease inhibitors (Pierce)] and homogenized onice in a tissue homogenizer (Omni International). The homogenate wascentrifuged at 1000×g, 5° C., for 10 min when pellet and supernatantwere separately collected. The supernatant was centrifuged for 1 h in aSW55 rotor at 140,000×g, 6° C. (Beckman LB80M Ultracentrifuge) and thepellet of this centrifugation was resuspended in ice cold PBS. Theresuspended pellet was re-centrifuged for 30 min at 30,000×g, 6° C. Thesupernatant was removed and the pellet was dissolved in solubilizationbuffer (1% Triton X-100 in 0.06M Tris-HCl, pH7.5, 0.001MNa-orthovanadate, 0.015M MgCl₂, cocktail of protease inhibitors) forsubsequent protein measurements, sododecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) followed by staining and immunoblotting asdescribed below for FIG. 7 . Prior to solubilization of the 30,000×gpellet a small amount was removed mechanically from the pellet,transferred and fixed in 2.5% glutaraldehyde in 0.113M Cacodylate buffer(pH7.4). After overnight fixation, the protein sample was processedthrough uranylacetate and osmium staining and embedded in Epon for TEMas described (Pytowsky et al., J Exp Med 167:421-439, 1988). Sections(60 nm) were examined and recorded in a Zeiss EM10. Photographs shownwere taken at Magn.×85,000.

Western Blot:

These procedures were used to obtain the data presented in FIG. 7 .Cells (2×10⁶) were grown in 75 cm² TCFs until ˜80% confluency when theywere placed on ice, washed with ice cold PBS, scraped and collected inPBS. The collected cell volume was divided into two equal parts. Onepart was used to obtain whole cell lysates while the second part wasused to prepare purified plasma membrane as described above and brieflysummarized as follows: To obtain purified plasma membrane, the cellswere centrifuged at 500×g, for 10 min and at 5° C. After removal of thesupernatant the cell pellet was resuspended in homogenization buffer[0.022M Na—PO₄, pH7.4, 0.001M MgCl₂, 0.25M sucrose, cocktail of proteaseinhibitors (Pierce)] and homogenized on ice in a tissue homogenizer(Omni International). The homogenate was centrifuged at 1000×g, 5° C.,for 10 min when pellet and supernatant were separately collected. Thesupernatant was centrifuged for 1 h in a SW55 rotor at 140,000×g, 6° C.(Beckman LB80M Ultracentrifuge) and the pellet of this centrifugationwas resuspended in ice cold PBS. The resuspended pellet wasre-centrifuged for 30 min at 30,000×g, 6° C. The supernatant was removedand the pellet was dissolved in solubilization buffer (1% Triton X-100in 0.06M Tris-HCl, pH7.5, 0.001M Na-Orthovanadate, 0.015M MgCl₂,cocktail of protease inhibitors) for subsequent protein measurements,sododecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followedby staining and immunoblotting as described below.

To obtain whole cell lysates, the cells in PBS were pelleted for 10 minat 250×g and 5° C., the supernatant was removed and the pelletresuspended in lysing buffer (2% Triton X-100 in 0.06M Tris-HCl, pH7.5,0.001M Na-orthovanadate, cocktail of protease inhibitors). The proteinconcentration was measured in the total cell lysates as well as theplasma membrane preparations. Equal amounts of proteins were preparedwith 2× Laemmli Sample Buffer under reducing conditions (5% 2-betamercaptoethanol, boiling for 3 min) and separated in linear 12% SDS-PAGEgels, electrophoretically transferred to nitrocellulose paper forimmunoblotting with antibodies (each at 0.005 mg/ml blocking buffer) toMDM-2, HDM-2 and p53. It should be noted, that the preparation of thetotal cell lysates and the lysates of the cells' plasma membrane for thetwo immunoblots followed exactly the procedure described above. Afterelectrophoretic transfer of the separated polypeptides, the remainingcharged sites of the nitrocellulose membrane were then blocked 2×30 minat RT with 5% dry milk in TBS-T buffer, washed with dH₂O and incubatedfor 1 h at Room Temperature (RT) with the primary antibody solution(MDM-2, HDM-2, p53). The membrane was then washed extensively with TBS-Tand reincubated for 30 min, RT, with the secondary antibody solution ofHRP-conjugated Donkey-anti-Mouse IgG (HRP-D anti-M IgG) 1:1000 in 0.1%milk in TBS-T). After extensive washing in TBS-T buffer followed by dH₂Oto remove non-reactants, Immun-Star HRP Peroxide Buffer+Immun-Star HRPLuminol/Enhancer (ratio 1:1) was added to the nitrocellulose membranesand the chemiluminescent reaction was exposed in complete darkness toX-ray film. Exposure time was 10 min.

Transmission Electron Microscopy:

Cells (1×10⁶) were grown in 6-well dishes overnight then spent mediumwas removed, the cells were washed with PBS and treated at 37° C. withPNC-28 at 50 μg/ml in PBS. At different time points (5-30 min) duringthe PNC-28 treatment the cells were washed and fixed in 3% bufferedparaformaldehyde supplemented with 0.01% glutaraldehyde for 1.5 h. Afterextensive washing, the cells were treated with glycine and NaBH₄ asdescribed above followed by 3 times washing in PBS. The cells were thenpost-fixed in 1% glutaraldehyde in cacodylate buffer (0.113M, pH7.2)overnight, at which time they were scraped into PBS and centrifuged intoa pellet. The cells were washed in cacodylate buffer and once morepost-fixed and stained in 1% osmium tetraoxide for 1 h, rt. The fixedcells were dehydrated through sequential passages in increasingconcentrations of ethanol and embedded in agar which was then exchangedfor Epon. After hardening the Epon for 72 h, thin sections (60 nm) werecut on an ultramicrotome, stained with uranyl acetate and examined in aZeiss EM10 transmission electron microscope.

Scanning Electron Microscopy:

Cells (3×10⁴) were grown on glass cover slips and treated with PNC-27(50 μg/ml) for 3 minutes. Cells were then fixed in 3% bufferedparaformaldehyde supplemented with 0.01% glutaraldehyde for 1.5 h. Afterinitial fixation, cover slips were rinsed several times with PBS for aminimum of 15 minutes, followed by post fixation with 1% osmiumtetroxide in 0.1M phosphate buffer, pH 7.4, for 1 hour. Cover slips werethen dehydrated using a series of graded ethyl alcohols (70% for 15 min,95% for 15 min. and 3 changes of 100% for 10 min. each). Cover slipswere then mounted on metal stubs, platinum sputter-coated and viewedusing a LEO 1550 scanning electron microscope.

Dose Response Experiments:

For the dose response experiments (n=3-5), 0.1 ml of each of the cells(70,000 cells/ml of culture medium) were seeded into each of the wellsof 96-well TCD (7000 cells/well) and allowed to adhere overnight. On thenext day, the medium was removed and 0.1 ml of the different PNC-27concentrations in PBS (37° C.) was added to triplicate wells and theincubation continued for 30 min at 37° C. At t=0 min and t=30 min atriplicate set of supernatants and cell lysates were removed that hadbeen incubated in PBS only. These two sets of samples were used toestablish background values for LDH release and LDH content of cellsincubated for 0 and 30 min, respectively, in PBS only. At the end of theincubation period 0.05 ml of culture supernatant were removed, and 0.05ml of lysis buffer (2% Triton-X-100 in dH₂O) were added to each well toobtain cell lysis and the release of cell-contained LDH. For themeasurement of the LDH activities present in the cell supernatants andthe cell lysates, 0.05 ml from each sample were mixed with 0.05 ml ofthe substrate mix (CytoTox 96 Non-radioactive Cytotoxicity Assay,Promega, Corp, Madison, WI) incubated in the dark and at roomtemperature for 30 min, when 0.05 ml of stop solution was added. Theresults were read at OD_(490nm) in an ELISA plate reader within 15 minof stopping the color development in the wells. The results werecalculated as advised by the manufacturers guidelines (CytoTox 96Non-radioactive Cytotoxicity Assay, Promega), and the data wereexpressed in % Cytotoxicity. Since for each dose (triplicatemeasurements), the values calculated for SEM were ±≤5% of the resultsshown, they do not appear in the computer-drawn graphs.

What is claimed is:
 1. A method of treating cancer in a subject, saidmethod comprising: providing a subject having a plurality of cancercells; and administering to the subject, a therapeutically effectiveamount of a composition including: an HDM-2 binding component selectedfrom the group consisting of:   12-26 p53 protein, residues(SEQ ID NO: 1) (PPLSQETFSDLWKLL), 17-26 p53 protein, residues(SEQ ID NO: 2) (ETFSDLWKLL), 12-26 p53 protein, residues (SEQ ID NO: 1)(P*P*LSQETFSDLWKLL), 17-26 p53 protein, residues (SEQ ID NO: 2)(E*T*FSDLWKLL), 12-26 p53 protein, residues (SEQ ID NO: 1)(P*P*L*SQETFSDLWKLL),

 and (SEQ ID NO. 10)   XFMXXXEXLX,

 where X in first position is actyl moiety (CHO), X in fourth positionis alpha-amino-isobutyric acid, X in fifth position isphosphonomethyl-phenylalanine, X in sixth position is6-chlorotryptophan, X in eighth position is1-amino-cyclopropanecarboxylic acid, X in tenth position is NH2; and amembrane resident component selected from the group consisting of:(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG, and (SEQ ID NO: 12)KKWKMRRNQFWVKVQRGLLR,

said membrane resident component bound to said HDM-2 binding component;wherein * denotes D-amino acid, and wherein selectively necrosing saidcancer cell, but does not affect the normal non-cancerous cells.
 2. Amethod of treating cancer in a subject, said method comprising:providing a subject having a plurality of cancer cells; andadministering to the subject, a therapeutically effective amount of acomposition including: an HDM-2 binding component selected from thegroup consisting of:   12-26 p53 protein, residues (SEQ ID NO: 1)(PPLSQETFSDLWKLL), 17-26 p53 protein, residues (SEQ ID NO: 2)(ETFSDLWKLL), 12-26 p53 protein, residues (SEQ ID NO: 1)(P*P*LSQETFSDLWKLL), 17-26 p53 protein, residues (SEQ ID NO: 2)(E*T*FSDLWKLL), 12-26 p53 protein, residues (SEQ ID NO: 1)(P*P*L*SQETFSDLWKLL), XFMXXXEXLX,

 where X in first position is actyl moiety (CHO), X in fourth positionis alpha-amino-isobutyric acid, X in fifth position isphosphonomethyl-phenylalanine, X in sixth position is6-chlorotryptophan, X in eighth position is1-amino-cyclopropanecarboxylic acid, X in tenth position is NH2 (SEQ IDNO. 10; and a membrane resident component selected from the groupconsisting of: (SEQ ID NO: 3)   K*K*WKMRRNQFWVKVQRG, (SEQ ID NO: 11)K*K*WKMRRNQFWVKVQRGLLR

 wherein X is actyl moiety (CHO), and

said membrane resident component bound to said HDM-2 binding component;wherein * denotes D-amino acid, and wherein selectively necrosing saidcancer cell, but does not affect the normal non-cancerous cells.
 3. Themethod of claim 1, further comprising the step of observing in a mediumof the cancer cells an early release of LDH.
 4. The method of claim 1,further comprising the step of observing membranolysis of said cancercells.
 5. The method of claim 1, further comprising the step ofobserving a decrease from a number of pretreatment cancer cells to anumber of post treatment cancer cells.
 6. The method of claim 1, furthercomprising the step of repeating the administering step until a resultis reached.
 7. The method of claim 1, further comprising the step ofobserving necrosis in the cancer cells.
 8. The method of claim 1,further comprising the step of observing a non-response in the normalcell, wherein the non-response indicates the normal cell is unaffected.9. The method of claim 1, wherein the administering step furthercomprises administering a PNC-27 (SEQ ID NO: 5) peptide, a PNC-28 (SEQID NO: 6) peptide, or combinations thereof.
 10. The method of claim 1,wherein the HDM-2 binding component is 12-26 p53 protein, residues(SEQ ID NO: 1)   (PPLSQETFSDLWKLL),


11. The method of claim 1, wherein the HDM-2 binding component is 17-26p53 protein, residues (SEQ ID NO: 2)   (ETFSDLWKLL),


12. The method of claim 1, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


13. The method of claim 10, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


14. The method of claim 11, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


15. The method of claim 2, wherein the HDM-2 binding component is 12-26p53 protein, residues (SEQ ID NO: 1)   (PPLSQETFSDLWKLL)


16. The method of claim 2, wherein the HDM-2 binding component is 17-26p53 protein, residues (SEQ ID NO: 2)   ETFSDLWKLL)


17. The method of claim 2, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


18. The method of claim 15, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


19. The method of claim 16, wherein the membrane resident component is(SEQ ID NO: 3)   KKWKMRRNQFWVKVQRG


20. The method of claim 1, wherein selective necrosis comprises poreformation.